We have used genetic methods to isolate mutant cell lines in which the activation of NFKappaB is constitutive. Complementation of these cells by overexpressing proteins that suppress the constitutive activation of NFKappaB has led to the functional identification of several novel candidate negative regulators. Constitutive activation of NFKappaB is common in many different cancers, where the ability of NFKappaB-regulated gene products to suppress apoptosis is important. We will investigate how different negative regulators control pathways that can lead to the activation of NFKappaB in response to many different cellular stresses by pursuing three specific aims: Specific Aim 1A: Validation of candidate negative regulators of NFKB and identification of new candidates. We will ablate the expression of candidate negative regulators in normal cells and test for consequent constitutive activation of NFKB. We will also test the effect of each candidate negative regulator in constitutive mutant cells that represent different complementation groups and that have biochemically distinct phenotypes. Also, we will test whether these proteins affect the induction of NFKappaB in response to IL-1 and TNF. To identify additional candidate negative regulators, we will complement additional constitutive mutants by using retroviral cDNA libraries and also by using Random Activation of Gene Expression, a novel technology that can drive high expression of any endogenous gene. Specific Aim 1B: Negative regulators of NFKappaB and cancer. We will determine the levels of expression of the novel negative regulators already in hand, and of additional ones to be isolated, in a variety of cancers in which NFKappaB is constitutively active, to identity tumor types in which loss of a particular regulator is frequent. We will then begin to explore the potential of using negative regulators as diagnostic aids and as possible targets for therapy. Specific Aim 2: Analysis of each validated negative regulator. A variety of assays will be employed to ascertain the physiological role of each validated negative regulator. We will analyze their effects on IKappaBalpha phosphorylation and on the activation of kinases already known to be involved in constitutive or induced NFKappaB activation. We will identify proteins associated with negative regulators by using co-immunoprecipitations, GST pull-downs and yeast two-hybrid analyses. We will also perform detailed structure-function analyses of negative regulators to help elucidate their mechanisms of action.